10) where R(pCa) represents the column of the measured ratios, and
8. To study cells at minimum pre-stress, two methods can be used
●
● Setting the final gap width to the average height of the cells.
●
● Zeroing the normal force of the rheometer with the measurement head of the rheometer lifted up in air. When bringing down the measurement head, compressed cells yield a positive normal force. Minimum average pre-stress is obtained with the cells at a gap width where the normal force is zero again.
Acknowledgments
We gratefully acknowledge financial support by Deutscher Akademischer Austauschdienst (DAAD), Germany, and the Ministry of Higher Education & Scientific Research, Iraq.
References
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nonlinear rheology of living cells. Annu Rev Mater Res 41:75–97
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fluids. J Non-Newtonian Fluid Mech 148:73–87
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Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_21, © Springer Science+Business Media LLC 2017
Chapter 21
Measurement of Cellular Behavior by Electrochemical Impedance Sensing
Simin ệz, Achim Breiling, and Christian Maercker
Abstract
There is a great demand for label-free in vitro assays in a high-throughput context, in order to measure cell viability and analyze cellular functions like cell migration or cell differentiation under noninvasive conditions. Here, we describe impedance measurement to quantify dynamic changes on cell morphology in real time. In order to monitor physiological changes, cells are grown in tissue culture vessels where gold electrodes are incorporated at the bottom. An alternating current signal of several kHz is applied to the electrodes and the resulting voltage is measured to calculate the cellular impedance. Since impedance is closely related to the area of the electrodes covered by the growing cells, parameters such as cell number, size of the cells attached to the electrodes, and cell-cell and cell-substrate/extracellular matrix interactions contribute to the overall impedance values.
Key words In vitro experiments, Cell based assays, Cell viability, Cell differentiation, Impedance sensing
1 Introduction
Electrochemical measurements in tissue culture to generate dielectric resistance profiles were first reported by the pioneering work of Giaever and Keese [1] and were further developed into a new field of biosensors now called electric cell-substrate impedance sensing (ECIS) [2]. Based on this principle, several systems evolved each equipped with its own specific features including sensors for pH, temperature, glucose, or oxygen-consumption [3, 4]. To monitor cell behavior electrically, cells are grown on special tissue culture vessels in which gold electrodes are incorporated at the bottom of the cultureware. An alternating current (AC) signal of several kHz is applied on the electrodes and the resulting voltage is measured to calculate the impedance; the AC equivalent to electric resistance, where the resulting output unit is in Ohms. As cells start to attach to the electrodes they serve mainly as insulators and restrict the current flow, thus leading to an increase in the impedance (Fig. 1).
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In general, impedance is closely related to the covered area of the electrodes and is thus mainly influenced by the cell number and size of the cells attached to the electrodes, but also cell-cell and cell-substrate/ECM interactions further contribute to the overall impedance values. Therefore, the impedance profiles, varying in magnitude and initial slopes depending on cell morphology and attachment of the cells, may reflect the cell type, cell state, and cell growth and this method may be used as a valuable tool to characterize the dynamics of cells in real time in a noninvasive manner [2].
A mathematical model was developed which determines three parameters describing impedance data (for details see [5, 6]).
Therein, the current flows beneath the cells are defined by the cell- substrate interaction, which mainly depends on the cell size and the space between the cells and the substratum. The spaces and formed junctions between the cells determine the current flow through the cell layer and define the barrier resistance (Rb).
Additionally, the membrane capacitance (Cm) can be included as well. Further interpretation of impedance data can be obtained if the phase of voltage is considered. The method discussed in this
Fig. 1 Principle of electrochemical measurement (kindly provided by Michael Angstmann)
Simin ệz et al.
chapter focuses on the measurement of impedance (Z) without regard to other parameters.
In order to monitor the dynamics of cell behavior, we use the ECIS system from Applied Biophysics where we describe measurements in 8-well plates (Fig. 2), which is suitable for functional analyses on a small scale, in particular to follow cell behavior, e.g., upon small molecule treatment or siRNA-mediated depletion of specific genes. Moreover, cells on the wells can be observed under the microscope. For high-throughput applications, e.g., viability tests in drug target validation assays, 96-well plates are recommended.
The ECIS system also allows more specialized assays, such as cell migration after electroporation (“wound healing”), or testing the endothelial barrier functions (trans-epithelial electrical resistance, TEER) with the TransFilter Adapter. However we will not be going over these techniques here [7, 8].
Another system which we tested but is not shown here is the xCELLigence real-time cell analysis (RTCA) system by ACEA Biosciences. The wells of the biochips of this system are not accessible by microscopy; however, this device is very convenient for high-throughput applications with up to five 96-well plates in one experiment. The RTCA system also is applicable for transmigration assays on specialized xCELLigence transwell plates [9].
2 Materials
1. Cell line: For most experiments, we used the human cell line NTERA 2 D1 (NT2, kind gift from Peter W. Andrews, University of Sheffield). The culturing conditions and medium mentioned below are specific for this cell line and have to be adjusted for each cell line.
2.1 Cell Culture
Fig. 2 ECIS 8W10E+ array (Applied Biophysics)
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